A novel method for quantification of gemcitabine and its metabolites 2',2'-difluorodeoxyuridine and gemcitabine triphosphate in tumour tissue by LC-MS/MS: comparison with (19)F NMR spectroscopy.

PURPOSE: To develop a sensitive analytical method to quantify gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolites 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) simultaneously from tumour tissue. METHODS: Pancreatic ductal adenocarcinoma tumour tissue from genetically engineered mouse models of pancreatic cancer (KP ( FL/FL ) C and KP ( R172H/+) C) was collected after dosing the mice with gemcitabine. (19)F NMR spectroscopy and LC-MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue. RESULTS: A (19)F NMR protocol was developed, which was capable of distinguishing the three analytes in tumour homogenates. However, it required at least 100 mg of the tissue in question and a long acquisition time per sample, making it impractical for use in large PK/PD studies or clinical trials. The LC-MS/MS protocol was developed using porous graphitic carbon to separate the analytes, enabling simultaneous detection of all three analytes from as little as 10 mg of tissue, with a sensitivity for dFdCTP of 0.2 ng/mg tissue. Multiple pieces of tissue from single tumours were analysed, showing little intra-tumour variation in the concentrations of dFdC or dFdU (both intra- and extra-cellular). Intra-tumoural variation was observed in the concentration of dFdCTP, an intra-cellular metabolite, which may reflect regions of different cellularity within a tumour. CONCLUSION: We have developed a sensitive LC-MS/MS method capable of quantifying gemcitabine, dFdU and dFdCTP in pancreatic tumour tissue. The requirement for only 10 mg of tissue enables this protocol to be used to analyse multiple areas from a single tumour and to spare tissue for additional pharmacodynamic assays

The Response of RIF-1 Fibrosarcomas to the Vascular-Disrupting Agent ZD6126 Assessed by In Vivo and Ex Vivo (1)H Magnetic Resonance Spectroscopy

The response of radiation-induced fibrosarcoma 1 (RIF-1) tumors treated with the vascular-disrupting agent (VDA) ZD6126 was assessed by in vivo and ex vivo (1)H magnetic resonance spectroscopy (MRS) methods. Tumors treated with 200 mg/kg ZD6126 showed a significant reduction in total choline (tCho) in vivo 24 hours after treatment, whereas control tumors showed a significant increase in tCho. This response was investigated further within both ex vivo unprocessed tumor tissues and tumor tissue metabolite extracts. Ex vivo high-resolution magic angle spinning (HRMAS) and (1)H MRS of metabolite extracts revealed a significant reduction in phosphocholine and glycerophosphocholine in biopsies of ZD6126-treated tumors, confirming in vivo tCho response. ZD6126-induced reduction in choline compounds is consistent with a reduction in cell membrane turnover associated with necrosis and cell death following disruption of the tumor vasculature. In vivo tumor tissue water diffusion and lactate measurements showed no significant changes in response to ZD6126. Spin-spin relaxation times (T(2)) of water and metabolites also remained unchanged. Noninvasive (1)H MRS measurement of tCho in vivo provides a potential biomarker of tumor response to VDAs in RIF-1 tumors

Antiretroviral therapy, fat redistribution and hyperlipidaemia in HIV-infected children in Europe. European Paediatric Lipodystrophy Group

Objectives: To estimate prevalence of body fat redistribution and dyslipidaemia in HIV-infected children and to assess associated risk factors, ultimately to inform the definition of lipodystrophy in children. Design: Cross-sectional observational study. Methods: During a 2-3 month period, 477 HIV-infected children aged ≥ 3 years (median 9.78; range, 3-18) in 30 paediatric HIV clinics were assessed at their first visit. Sociodemographic, clinical and immunological data were recorded and the presence or absence of clinical signs of fat redistribution (peripheral lipoatrophy and central lipohypertrophy) determined according to an agreed protocol. Laboratory indicators of lipid/glucose metabolism were recorded for all children in 18 centres. Results: Prevalence was 26.0% [95% confidence interval (CI), 22.1-30.2] for any fat redistribution, 8.81% (95% CI, 6.42-11.7) for central lipohypertrophy, 7.55% (95% CI, 5.34-10.3) for peripheral lipoatrophy and 9.64% (95% CI, 7.15-12.7) for the combined subtype (more than one sign of each). Independent predictors of fat redistribution included Centers for Disease Control and Prevention class C disease, female gender, ever used versus never use of protease inhibitors and of stavudine. Increasing time since initiation of antiretroviral therapy was associated with increased severity of fat redistribution. In the metabolic assessment subgroup, 27% (95% CI, 21.6-32.7) of children had hypercholesterolaemia and 21% (95% CI, 16.4-26.6) hypertriglyceridaemia; however, significantly more children had fat redistribution in this subgroup than overall (31%). Conclusions: Approximately a quarter of children and adolescents could be taken to have signs of lipodystrophy, with clinical presentation and risk factors similar to those described in adults

Long-term clinical effects of a chlorhexidine varnish implemented treatment strategy for chronic periodontitis

Background: Scaling and root planing in combination with oral hygiene monitoring are still considered the therapeutic standards for periodontitis. Although this treatment concept customarily results in satisfactory clinical improvements, treatment outcome may become less favorable predominantly when full access to periodontal defects is compromised, thereby leaving accretions behind. The purpose of this study was to investigate, over a 9-month period, the clinical benefits of a treatment strategy for chronic periodontitis based on a combination of sequential scaling and root planing and subgingival chlorhexidine varnish administration. Methods: This randomized controlled, single blind, parallel trial included 26 volunteers with chronic periodontitis. The control group received oral hygiene instructions and was scaled and root planed in two sessions. The test group received the same instructions and treatment; however, all pockets were additionally disinfected using a highly concentrated chlorhexidine varnish. Clinical response parameters were recorded at baseline and at 1, 3, 6, and 9 months. The impact of the initial strategy on the decision-making process for supplementary therapy at 9 months was investigated based on treatment decisions made by five independent clinicians. Results: Both treatment strategies showed significant reductions in probing depth and gains in clinical attachment at study termination in comparison with baseline (P = 7 mm) around multirooted teeth seemed to benefit most from the combination strategy, resulting in an additive pocket reduction of 1.06 mm (P= 0.009) and a clinical attachment gain of 0.54 mm (P = 0.048) in comparison to scaling and root planing alone. A trend toward a reduction of surgical treatment needs following the varnish-implemented strategy was found (P= 0.076). Conclusion: These findings suggest that the outcome of initial periodontal therapy may benefit from the adjunctive subgingival administration of a highly concentrated chlorhexidine varnish

Human immunodeficiency virus type 1 infection and breast milk. The Italian Register for HIV Infection in Children.

Major questions are whether mothers infected with the Human Immunodeficiency Virus type 1 (HIV-1) transmit the virus through breast milk and the magnitude of the additional transmission risk. The demonstration of a dose-response effect is an epidemiological method to demonstrate causality. Thus, a study was carried out by the Italian Register for HIV Infection in Children on 961 children of known infection status. Duration of breast-feeding was considered as the level of exposure in 168 ever breast-fed children. Results showed that duration of practice significantly increased the risk of transmission. The adjusted infection odds ratio for one day of breast- versus exclusive formula-feeding was 1.19 with narrow confidence limits (1.10-1.28). In a second study by the Register on 556 children of known infection status and derived prospectively, an infection odds ratio of 2.55 (confidence interval: 1.03-6.37) was calculated in breast- versus exclusively formula-fed children. Several lines of evidence, including the above-mentioned data from the Italian Register for HIV Infection in Children, showed a contribution of breast-feeding to mother-to-child HIV-1 transmission. Thus, this practice is now discouraged in HIV-1 infected mothers living in industrialized societies where formula feeding is practical and attainable. Mode of feeding was known in 2183 children enrolled in the Register and born to HIV-1 infected mothers since 1981. It could be observed that feeding habits of at-risk infants changed in Italy in the middle 1980s, when a large majority of subjects was identified at birth